Sample treatment method for detecting macrolide antibiotics in plant source matrix sample

Abstract

The invention relates to the technical field of antibiotic detection, and particularly discloses a sample treatment method for detecting macrolide antibiotics in a plant source matrix sample. The sample treatment method provided by the invention comprises the following steps: uniformly mixing a plant source matrix sample with a buffer solution, adding acidified acetonitrile, salting out and absorbing water by using a mixed inorganic salt, and purifying by using an adsorbent comprising anhydrous magnesium sulfate, an aliphatic diamine derivative and O-terminated octadecyl bonded silica gel to obtain the treated plant source matrix sample. Through the systematic design of extraction-separation-purification, the chemical properties of the target object and the characteristics of the plant matrix are combined, and the synergistic effect of the buffer solution, acidified acetonitrile, salting-out and multiple adsorbents is utilized, so that matrix interference can be effectively removed, the target object is stable, and efficient extraction of macrolide antibiotics is realized. The method is suitable for detecting the macrolide antibiotics such as lincomycin, tilmicosin and erythromycin in the plant source matrix.

Description

Technical Field

[0001] The present invention relates to the technical field of antibiotic detection, in particular to a sample processing method for detecting macrolide antibiotics in a plant-derived matrix sample. Background Art

[0002] With the large-scale development of animal husbandry and agriculture worldwide, antibiotic use has increased annually, and excessive antibiotic use in animal husbandry is widespread. Macrolide antibiotics, a class of antimicrobial drugs containing a 12-16 carbon lactone ring in their molecular structure, are fast-acting antibacterial agents and account for a large proportion of overall antibiotic use. These drugs are not fully metabolized in animals, and residual antibiotics can enter the aquatic environment through animal excretion. During the cultivation of crops (such as fruits, vegetables, and Chinese herbal medicines), antibiotics carried in irrigation water can be concentrated in the plants through root absorption and other pathways, resulting in trace antibiotic residues in plant-based samples. However, traditional antibiotic detection techniques for livestock samples primarily target animal tissue matrices, and their extraction and cleanup methods struggle to effectively adapt to the complex matrix environments of plant-based samples. Plant samples often contain a large number of polar interfering substances (such as organic acids, phenolic compounds, and carbohydrates), which, when present with antibiotics, can affect detection accuracy and sensitivity. Directly employing existing technologies for detection requires cumbersome pretreatment steps such as multiple liquid-liquid extractions, multi-step solid-phase cleanup, or repeated chromatographic separations. These processes are not only time-consuming and complex, but can also result in the loss or contamination of the target antibiotic, severely limiting detection efficiency.

[0003] While high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) has become a mainstream method for detecting trace pollutants, its application in plant-based samples is limited by the lag in pretreatment technology. Developing efficient and simple pretreatment methods tailored to the specific characteristics of plant matrices to accurately extract and purify trace antibiotics, and then accurately measure them using liquid chromatography-mass spectrometry, remains a pressing technical challenge in the detection of plant-derived antibiotic residues. Summary of the Invention

[0004] To address the challenges of existing methods for detecting macrolide antibiotics in plant-derived matrix samples, including complex sample pretreatment steps, cumbersome procedures, time-consuming processes, and low extraction efficiency, the present invention provides a method for detecting macrolide antibiotics in plant-derived matrix samples and a sample treatment method. This sample treatment method can rapidly and effectively extract macrolide antibiotics from the plant-derived matrix, providing highly clean test samples for subsequent precise detection using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS).

[0005] In order to solve the above technical problems, the technical solution provided by the present invention is: In a first aspect, the present invention provides a sample processing method for detecting macrolide antibiotics in a plant-derived matrix sample, the sample processing method comprising the following steps: The plant-derived matrix sample is mixed with a buffer solution, acidified acetonitrile is added for extraction, and then mixed inorganic salts are added to the extract, solid-liquid separation is performed, and the resulting liquid phase is added to an adsorbent for adsorption, solid-liquid separation is performed again, and the liquid phase is obtained to obtain the treated plant-derived matrix sample; The adsorbent includes anhydrous magnesium sulfate, aliphatic diamine derivatives and O-terminated octadecyl bonded silica gel.

[0006] The present invention provides a sample preparation method for detecting macrolide antibiotics in plant-derived matrix samples. Soaking the sample in a buffer solution removes most of the unwanted matrix, reduces interference from other macromolecules, and allows the analyte to form a stable complex. Acidified acetonitrile improves extraction efficiency, inhibits hydrolysis of the target compound, and facilitates subsequent protein removal. The addition of mixed inorganic salts shortens solid-liquid separation time and improves extraction efficiency through salting out. The water absorption of the mixed inorganic salts also prevents moisture from affecting subsequent adsorbent performance, improving detection stability. The anhydrous magnesium sulfate, aliphatic diamine derivative, and O-terminated octadecyl bonded silica gel (C18) in the adsorbent each act to target different types of impurities, forming a multi-step purification system. This sample preparation method ultimately achieves efficient extraction of macrolide antibiotics, removes matrix interference, and stabilizes the target compound. This invention provides a reliable pretreatment solution for the detection of trace residues of macrolide antibiotics and is particularly suitable for high-throughput analysis of multi-component macrolide antibiotics in complex plant-derived samples.

[0007] The present invention achieves efficient extraction of macrolide antibiotics through a systematic "extraction-separation-purification" design, combines the chemical properties of the target with the characteristics of the plant matrix, and utilizes the synergistic effects of buffer solution, acidified acetonitrile, salting out, and adsorbent. It can also effectively remove matrix interference and stabilize the target, providing a reliable pretreatment solution for the detection of trace residual macrolide antibiotics.

[0008] Acetonitrile is a polar organic solvent with good solubility for macrolide antibiotics. Acidified acetonitrile can adjust the acidity of the system, protonating the amino groups of the target compounds to form positively charged ions, thereby enhancing their interaction with acetonitrile (such as ion-dipole interaction) and further improving the extraction efficiency.

[0009] Anhydrous magnesium sulfate in the adsorbent continuously dehydrates and further removes residual water, preventing aqueous impurities (such as water-soluble sugars and amino acids) from entering subsequent detection steps. It also maintains a dry system, preventing degradation or adsorption loss of the target compound due to the presence of water. Aliphatic diamine derivatives can remove acidic impurities. They adsorb acidic substances (such as organic acids, phenolic acids, and nucleic acid fragments) in plant matrices through ion exchange or hydrogen bonding. Macrolide antibiotics are positively charged under acidic conditions, while acidic impurities are negatively charged. Diamine derivatives selectively adsorb negatively charged impurities, reducing their interference with the target compound. O-terminated octadecyl bonded silica gel (C18) removes non-polar impurities and adsorbs non-polar impurities such as lipids, pigments, and sterols through hydrophobic interactions. Plant-derived matrices often contain a large amount of fat-soluble components (such as chlorophyll and carotenoids). C18 can effectively remove these impurities, preventing peak broadening, tailing, or ion suppression in chromatographic analysis, thereby improving detection sensitivity and chromatographic separation.

[0010] Preferably, the mass ratio of the anhydrous magnesium sulfate, the aliphatic diamine derivative and the O-terminated octadecyl bonded silica gel is 16-20:0.8-1.2:0.8-1.2.

[0011] Preferably, the mass ratio of the plant-derived matrix sample to the adsorbent is 0.4-0.6:1.8-2.2.

[0012] Preferably, the macrolide antibiotics include at least one of lincomycin, tilmicosin, roxithromycin or erythromycin.

[0013] Preferably, the buffer solution comprises a Mcllvaine buffer solution with a pH of 3.8-4.2.

[0014] Preferably, the mixed inorganic salt comprises a strong acid salt and anhydrous sulfate.

[0015] Preferably, the mass ratio of the strong acid salt to the anhydrous sulfate is 1.5-2.5:3.5-4.5.

[0016] Preferably, the strong acid salt comprises sodium chloride, and the anhydrous sulfate salt comprises anhydrous magnesium sulfate.

[0017] Preferably, the aliphatic diamine derivative includes N-propylethylenediamine.

[0018] Preferably, the volume ratio of the organic weak acid to acetonitrile in the acidified acetonitrile is 0.08-0.12:99.88-99.92.

[0019] Illustratively, the organic weak acid is acetic acid.

[0020] Preferably, after the adsorbent is added for adsorption, the solid and liquid are separated, the obtained liquid phase is dried, and then a formic acid aqueous solution-methanol mixture is added for redissolution, and the sample is filtered to obtain a treated plant-derived matrix sample.

[0021] Preferably, the reconstituted solution is a formic acid aqueous solution-methanol mixture, wherein the volume proportion of formic acid in the formic acid aqueous solution is 0.18%-2.4%.

[0022] Preferably, the volume ratio of the aqueous formic acid solution to methanol is (7.8-8.2): (1.8-2.2).

[0023] For example, the reconstitution solution is prepared by mixing a formic acid aqueous solution with a volume concentration of 0.2% and methanol in a volume ratio of 8:2.

[0024] Preferably, the plant-derived matrix sample includes at least one of honeysuckle or chrysanthemum.

[0025] Honeysuckle and chrysanthemum matrices contain flavonoids (such as chlorogenic acid and luteolin), terpenes, polysaccharides, mucilage, and volatile oils. Polar polyphenols easily form hydrogen bonds or ionic interactions with antibiotics, affecting extraction efficiency; polysaccharides can cause the extract to become viscous and clog the chromatographic column; and volatile oils can interfere with mass spectrometry ionization. The sample preparation method for detecting macrolide antibiotics in plant-derived matrix samples provided herein is applicable to the detection of macrolide antibiotics in matrices such as honeysuckle and chrysanthemum.

[0026] The treated samples obtained using the sample treatment method of the present invention can effectively overcome interference from other substances in the plant-derived matrix with the detection of macrolide antibiotics. The treatment method is simple and rapid to operate, and its detection results are accurate. It can be widely promoted and applied, and is particularly suitable for the detection of macrolide antibiotics in complex matrices such as honeysuckle or chrysanthemum.

[0027] The treated plant-derived matrix sample obtained based on the sample processing method provided above is used as a test sample, and the macrolide antibiotics in the test sample can be detected by liquid chromatography-mass spectrometry detection method.

[0028] Using roxithromycin as the internal standard, samples treated with the sample treatment method provided by the present invention on plant-derived matrices such as honeysuckle or chrysanthemum were used as test samples. Liquid chromatography-mass spectrometry was used to detect macrolide antibiotics such as lincomycin, tilmicosin, and erythromycin. The detection method demonstrated high sensitivity and good repeatability, with spiked recoveries ranging from 81.51% to 109.03% and a detection limit as low as 0.5 μg / kg. The experimental results fully demonstrated the feasibility and scientific nature of the sample treatment method provided by the present invention, providing a reliable pretreatment technology solution for the detection of macrolide antibiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the characteristic ion chromatogram of the lincomycin standard in the verification example of the present invention, wherein: Figure 1 A) Representative characteristic ion mass chromatogram of lincomycin standard. Figure 1 B) Represents the characteristic ion accuracy ratio of lincomycin standard. Figure 1 C) Characteristic daughter ion fragmentation pattern of lincomycin standard; Figure 2 is the characteristic ion chromatogram of the tilmicosin standard in the verification example of the present invention, wherein: Figure 2 A) Characteristic ion mass chromatogram of tilmicosin standard. Figure 2 B) Represents the characteristic ion accuracy ratio of tilmicosin standard. Figure 2 C) Characteristic daughter ion fragmentation pattern of tilmicosin standard; Figure 3 This is the characteristic ion chromatogram of the erythromycin standard in the verification example of the present invention, wherein: Figure 3 A) Representative characteristic ion mass chromatogram of erythromycin standard. Figure 3 B) Represents the characteristic ion accuracy ratio of erythromycin standard. Figure 3 C) Characteristic daughter ion fragmentation pattern of erythromycin standard; Figure 4 This is the characteristic ion chromatogram of the roxithromycin standard in the verification example of the present invention, wherein: Figure 4 A) Representative characteristic ion mass chromatogram of roxithromycin standard. Figure 4 B) Represents the characteristic ion accuracy ratio of roxithromycin standard. Figure 4 C) Characteristic daughter ion fragmentation pattern of roxithromycin standard; Figure 5 The standard curve of lincomycin in Example 1 of the present invention; Figure 6 The standard curve of tilmicosin in Example 1 of the present invention; Figure 7 The standard curve of erythromycin in Example 1 of the present invention; Figure 8 The liquid chromatogram of the test solution 1-50 in Example 4 of the present invention; Figure 9 This is the liquid chromatogram of the test solution 2-50 in Effect Example 4 of the present invention. DETAILED DESCRIPTION

[0030] The technical solution of the present invention will be clearly and completely described below. It should be understood that the specific embodiments described here can enable those skilled in the art to understand the present invention more comprehensively, but the present invention is not limited to the scope of the described embodiments.

[0031] The water in the embodiment of the present invention is the first-class water specified in GB / T6682; N-propylethylenediamine (PSA), model: Cleanert PSA; particle size: 40-60 μm; O-terminated octadecyl bonded silica gel (C18), model: Cleanert S C18, particle size: 50 μm 60A; Uncapped octadecyl bonded silica gel (C18), model: Cleanert ODS C18, particle size: 50 μm 60A; Graphite carbon, model: Cleanert PestiCarb, particle size: 120-400 mesh; Neutral alumina, particle size: 100-300 mesh, 75%, pH value: 7.5±0.5; Acidic alumina, particle size: 100-300 mesh, pH value: 3.8-4.8; Lincomycin standard, purity 99.7%, was purchased from Beijing Coast Hongmeng Standard Material Technology Co., Ltd.; Tilmicosin standard, purity 99.2%, was purchased from Beijing Manhag Biotechnology Co., Ltd.; Roxithromycin standard, purity 98.7%, was purchased from Beijing Manhag Biotechnology Co., Ltd.; Erythromycin standard, with a purity of 99.8%, was purchased from Beijing Manhag Biotechnology Co., Ltd.

[0032] It should be noted that roxithromycin was used as the internal standard when determining antibiotics in this invention. The internal standard is added to the sample processing method "(3) Processing of Plant-derived Matrix Samples" provided in the Examples for evaluating the detection method and is not a necessary step in sample processing. If the purpose is to process the sample, the internal standard can be omitted or other macrolide antibiotics can be added as the internal standard.

[0033] Example 1 The present invention provides a sample processing method for detecting macrolide antibiotics in a plant-derived matrix sample. The sample processing method comprises the following steps: (1) Preparation of basic solution Preparation of Mcllvaine buffer solution: Take 1000 mL of 0.1 mol / L citric acid solution and 625 mL of 0.2 mol / L Na2HPO4 solution, mix well, and adjust to pH 4.0 with 0.1 mol / L hydrochloric acid solution; Acidify acetonitrile: dilute 1 mL of acetic acid to 1000 mL with acetonitrile.

[0034] (2) Preparation of mixed inorganic salts and adsorbents Take 2g NaCl and 4g anhydrous MgSO4 and mix them evenly to obtain a mixed inorganic salt; Take 1.8 g of anhydrous MgSO4, 0.1 g of N-propylethylenediamine (PSA) and 0.1 g of O-terminated octadecyl bonded silica gel, mix them evenly, and obtain an adsorbent.

[0035] (3) Processing of plant-derived matrix samples Weigh 0.5 g (accurate to 0.01 g) of honeysuckle and place it in a 50 mL stoppered centrifuge tube. Add 100 μL of roxithromycin diluted to 1 μg / mL as an internal standard, then add 5 mL of Mcllvaine buffer solution. Place in the dark and let it stand for 30 min. Then add 20 mL of acidified acetonitrile and vortex at 2500 rpm on a multitube vortexer for 30 min. After the vortexing, mixed inorganic salts were added to the resulting solution, mixed thoroughly, and centrifuged at 6000 rpm for 5 min to obtain the first supernatant; The first supernatant was transferred to a 50 mL stoppered centrifuge tube containing the above-mentioned adsorbent, vortexed thoroughly, and centrifuged at 6000 rpm for 5 min to obtain the second supernatant; The second supernatant was taken and dried with nitrogen in a 45°C water bath. 1.0 mL of the reconstitution solution was added and vortexed for 1 min to dissolve the residue. The solution was filtered through a 0.22 μm organic filter membrane to obtain the treated sample solution, which was recorded as test solution 1.

[0036] Example 2 The present invention provides a sample processing method for detecting macrolide antibiotics in a plant-derived matrix sample. The sample processing method comprises the following steps: (1) Preparation of basic solution Preparation of Mcllvaine buffer solution: Take 1000 mL of 0.1 mol / L citric acid solution and 625 mL of 0.2 mol / L Na2HPO4 solution, mix well, and adjust to pH 4.0 with 0.1 mol / L hydrochloric acid solution; Acidify acetonitrile: dilute 1 mL of acetic acid to 1000 mL with acetonitrile.

[0037] (2) Preparation of mixed inorganic salts and adsorbents Take 2g NaCl and 4g anhydrous MgSO4 and mix them evenly to obtain a mixed inorganic salt; Take 1.8 g of anhydrous MgSO4, 0.1 g of N-propylethylenediamine (PSA) and 0.1 g of O-terminated octadecyl bonded silica gel, mix them evenly, and obtain an adsorbent.

[0038] (3) Processing of plant-derived matrix samples Weigh 0.5 g (accurate to 0.01 g) of chrysanthemum and place it in a 50 mL stoppered centrifuge tube. Add 100 μL of roxithromycin diluted to 1 μg / mL as an internal standard, then add 5 mL of Mcllvaine buffer solution. Place in the dark and let it stand for 30 min. Then add 20 mL of acidified acetonitrile and vortex at 2500 rpm on a multitube vortexer for 30 min. After the vortexing, mixed inorganic salts were added to the resulting solution, mixed thoroughly, and centrifuged at 6000 rpm for 5 min to obtain the first supernatant; The first supernatant was transferred to a 50 mL stoppered centrifuge tube containing the above-mentioned adsorbent, vortexed thoroughly, and centrifuged at 6000 rpm for 5 min to obtain the second supernatant; The second supernatant was taken and dried with nitrogen in a 45°C water bath. 1.0 mL of the reconstitution solution was added and vortexed for 1 min to dissolve the residue. The solution was filtered through a 0.22 μm organic filter membrane to obtain the treated sample solution, which was recorded as test solution 2.

[0039] Example 3 The present invention provides a sample processing method for detecting macrolide antibiotics in a plant-derived matrix sample. The sample processing method comprises the following steps: (1) Preparation of basic solution Preparation of Mcllvaine buffer solution: Take 1000 mL of 0.1 mol / L citric acid solution and 625 mL of 0.2 mol / L Na2HPO4 solution, mix well, and adjust to pH 3.8 with 0.1 mol / L hydrochloric acid solution; Acidify acetonitrile: dilute 1 mL of acetic acid to 1000 mL with acetonitrile.

[0040] (2) Preparation of mixed inorganic salts and adsorbents Take 1.5g NaCl and 3.5g anhydrous MgSO4 and mix them evenly to obtain a mixed inorganic salt; Take 1.6 g of anhydrous MgSO4, 0.12 g of N-propylethylenediamine (PSA) and 0.08 g of O-terminated octadecyl bonded silica gel, mix them evenly, and obtain an adsorbent.

[0041] (3) Processing of plant-derived matrix samples Weigh 0.4 g (accurate to 0.01 g) of honeysuckle and place it in a 50 mL stoppered centrifuge tube. Add 100 μL of roxithromycin diluted to 1 μg / mL as an internal standard, then add 5 mL of Mcllvaine buffer solution. Place in the dark and let it stand for 30 min. Then add 20 mL of acidified acetonitrile and vortex at 2500 rpm on a multitube vortexer for 30 min. After the vortexing, mixed inorganic salts were added to the resulting solution, mixed thoroughly, and centrifuged at 6000 rpm for 5 min to obtain the first supernatant; The first supernatant was transferred to a 50 mL stoppered centrifuge tube containing the above-mentioned adsorbent, vortexed thoroughly, and centrifuged at 6000 rpm for 5 min to obtain the second supernatant; The second supernatant was taken and dried with nitrogen in a 45°C water bath. 1.0 mL of the reconstitution solution was added and vortexed for 1 min to dissolve the residue. The solution was filtered through a 0.22 μm organic filter membrane to obtain the treated sample solution, which was recorded as test solution 3.

[0042] Example 4 The present invention provides a sample processing method for detecting macrolide antibiotics in a plant-derived matrix sample. The sample processing method comprises the following steps: (1) Preparation of basic solution Preparation of Mcllvaine buffer solution: Take 1000 mL of 0.1 mol / L citric acid solution and 625 mL of 0.2 mol / L Na2HPO4 solution, mix well, and adjust to pH 4.2 with 0.1 mol / L hydrochloric acid solution; Acidify acetonitrile: dilute 1 mL of acetic acid to 1000 mL with acetonitrile.

[0043] (2) Preparation of mixed inorganic salts and adsorbents Take 2.5g NaCl and 4.5g anhydrous MgSO4 and mix them evenly to obtain a mixed inorganic salt; Take 2.0 g of anhydrous MgSO4, 0.08 g of N-propylethylenediamine (PSA) and 0.12 g of O-terminated octadecyl bonded silica gel, mix them evenly, and obtain an adsorbent.

[0044] (3) Processing of plant-derived matrix samples Weigh 0.6 g (accurate to 0.01 g) of honeysuckle and place it in a 50 mL stoppered centrifuge tube. Add 100 μL of roxithromycin diluted to 1 μg / mL as an internal standard, then add 5 mL of Mcllvaine buffer solution. Place in the dark and let it stand for 30 min. Then add 20 mL of acidified acetonitrile and vortex at 2500 rpm on a multitube vortexer for 30 min. After the vortexing, mixed inorganic salts were added to the resulting solution, mixed thoroughly, and centrifuged at 6000 rpm for 5 min to obtain the first supernatant; The first supernatant was transferred to a 50 mL stoppered centrifuge tube containing the above-mentioned adsorbent, vortexed thoroughly, and centrifuged at 6000 rpm for 5 min to obtain the second supernatant; The second supernatant was taken and dried with nitrogen in a 45°C water bath. 1.0 mL of the reconstitution solution was added and vortexed for 1 min to dissolve the residue. The solution was filtered through a 0.22 μm organic filter membrane to obtain the treated sample solution, which was recorded as test solution 4.

[0045] Comparative Example 1 The comparative example of the present invention provides a sample processing method for detecting macrolide antibiotics in a plant-derived matrix sample. The sample processing method is basically the same as that in Example 1, except that: when preparing the adsorbent, only the "O-capped octadecyl bonded silica gel" is replaced with an equal amount of "ODS C18", that is, it is replaced with uncapped octadecyl bonded silica gel. The remaining steps and parameters are the same as those in Example 1, and a test solution is finally prepared, which is recorded as test solution pair 1.

[0046] Comparative Example 2 The comparative example of the present invention provides a sample processing method for detecting macrolide antibiotics in a plant-derived matrix sample. The sample processing method is basically the same as that in Example 2, except that: when preparing the adsorbent, only the "O-blocked octadecyl bonded silica gel" is replaced with an equal amount of "ODS C18", that is, it is replaced with unblocked octadecyl bonded silica gel. The remaining steps and parameters are the same as those in Example 2, and finally a test solution is prepared, which is recorded as test solution pair 2.

[0047] Comparative Example 3 The comparative example of the present invention provides a sample processing method for detecting macrolide antibiotics in a plant-derived matrix sample. The sample processing method is basically the same as that in Example 1, except that: when preparing the adsorbent, only "N-propylethylenediamine" is replaced with an equal amount of "graphite carbon", and the remaining steps and parameters are the same as those in Example 1. Finally, a test solution is prepared, which is recorded as test solution pair 3.

[0048] Comparative Example 4 The comparative example of the present invention provides a sample processing method for detecting macrolide antibiotics in a plant-derived matrix sample. The sample processing method is basically the same as that in Example 2, except that: when preparing the adsorbent, only "N-propylethylenediamine" is replaced with an equal amount of "graphite carbon", and the remaining steps and parameters are the same as those in Example 2. Finally, a test solution is prepared, which is recorded as test solution pair 4.

[0049] Comparative Example 5 The comparative example of the present invention provides a sample processing method for detecting macrolide antibiotics in a plant-derived matrix sample. The sample processing method is basically the same as that in Example 1, except that: when preparing the adsorbent, only "N-propylethylenediamine" is replaced with an equal amount of "neutral alumina", and the remaining steps and parameters are the same as those in Example 1. Finally, a test solution is prepared, which is recorded as test solution pair 5.

[0050] Comparative Example 6 The comparative example of the present invention provides a sample processing method for detecting macrolide antibiotics in a plant-derived matrix sample. The sample processing method is basically the same as that in Example 2, except that: when preparing the adsorbent, only "N-propylethylenediamine" is replaced with an equal amount of "neutral alumina", and the remaining steps and parameters are the same as those in Example 2. Finally, a test solution is prepared, which is recorded as test solution pair 6.

[0051] Comparative Example 7 The comparative example of the present invention provides a sample processing method for detecting macrolide antibiotics in a plant-derived matrix sample. The sample processing method is basically the same as that in Example 1, except that: when preparing the adsorbent, only "N-propylethylenediamine" is replaced with an equal amount of "acidic alumina", and the remaining steps and parameters are the same as those in Example 1. Finally, a test solution is prepared, which is recorded as test solution pair 7.

[0052] Comparative Example 8 The comparative example of the present invention provides a sample processing method for detecting macrolide antibiotics in a plant-derived matrix sample. The sample processing method is basically the same as that in Example 2, except that: when preparing the adsorbent, only "N-propylethylenediamine" is replaced with an equal amount of "acidic alumina", and the remaining steps and parameters are the same as those in Example 2. Finally, a test solution is prepared, which is recorded as test solution pair 8.

[0053] Comparative Example 9 The comparative example of the present invention provides a sample processing method for detecting macrolide antibiotics in a plant-derived matrix sample. The sample processing method is basically the same as that in Example 1, except that: (1) when preparing the mixed inorganic salt, only "anhydrous magnesium sulfate" is replaced by an equal amount of "anhydrous sodium sulfate"; (2) when preparing the adsorbent, only "anhydrous magnesium sulfate" is replaced by an equal amount of "anhydrous sodium sulfate"; (3) 20, 50 and 100 μL of a mixed standard of lincomycin, tilmicosin and erythromycin with a concentration of 1 μg / mL are added respectively; the remaining steps and parameters are the same as those in Example 1, and finally, 9 series of test solutions are prepared, namely, test solution pair 1-20, test solution pair 1-50 and test solution pair 1-100.

[0054] Comparative Example 10 The comparative example of the present invention provides a sample processing method for detecting macrolide antibiotics in a plant-derived matrix sample. The sample processing method is basically the same as that in Example 1, except that: (1) when preparing the mixed inorganic salt, only "anhydrous magnesium sulfate" is replaced by an equal amount of "anhydrous sodium sulfate"; (2) when preparing the adsorbent, only "anhydrous magnesium sulfate" is replaced by an equal amount of "anhydrous sodium sulfate"; (3) 20, 50 and 100 μL of a mixed standard of lincomycin, tilmicosin and erythromycin with a concentration of 1 μg / mL are added respectively; the remaining steps and parameters are the same as those in Example 2, and finally, the test solutions are prepared, which are recorded as 10 series of test solutions, namely, test solution pair 2-20, test solution pair 2-50 and test solution pair 2-100.

[0055] Verification Example Based on the above-mentioned sample processing method for detecting macrolide antibiotics in plant-derived matrix samples, the present invention provides a method for detecting macrolide antibiotics in plant-derived matrix samples, comprising the following steps: (1) Preparation of standard solution Standard stock solution: Take an appropriate amount of each standard into a 10 mL volumetric flask, dissolve it in methanol and dilute to the scale to prepare a standard stock solution with a concentration of approximately 1 mg / mL.

[0056] Standard internal standard working solution: Dispense 0.1 mL of 1 mg / mL roxithromycin standard stock solution into a 10 mL volumetric flask and dilute to the mark with methanol to prepare a 10 μg / mL standard internal standard working solution. During the experiment, dilute to 1 μg / mL with methanol.

[0057] Mixed standard working solution: Prepare a 10 μg / mL mixed standard working solution by adding 0.1 mL of each of the lincomycin, tilmicosin, and erythromycin stock solutions (1 mg / mL) to the same volumetric flask. Add methanol and dilute to the mark. During the experiment, dilute to 1 μg / mL with methanol.

[0058] (2) Detection The test solutions, standard internal standard working solutions and mixed standard working solutions of the examples and comparative examples were injected into a high performance liquid chromatography-mass spectrometer, respectively. The chromatograms were recorded, and the contents of lincomycin, tilmicosin, erythromycin and roxithromycin and the spiked recovery rates were calculated.

[0059] The HPLC conditions are as follows: Chromatographic column: Intersil C 18 Chromatographic column (150 mm × 2.1 mm, 3 μm); Mobile phase A: 0.1% formic acid in water; Mobile phase B: acetonitrile; Flow rate: 0.3 mL / min; Detection wavelength: 270nm; Column temperature: 27°C; Injection volume: 10 μL; Sample pan temperature: 10°C.

[0060] The elution program was gradient elution, and the elution program was shown in Table 1.

[0061] Table 1

[0062] The retention times of lincomycin, tilmicosin, erythromycin, and roxithromycin are shown in Table 2.

[0063] Table 2

[0064] The mass spectrometry conditions are as follows: Ion source: electrospray ion source; Scanning mode: positive ion scanning; Detection method: Multiple reaction monitoring; Spray voltage: 5500V; Atomizing gas pressure: 0.069MPa; Air curtain pressure: 0.69MPa; Ion source temperature: 350°C; The parameters of the reaction qualitative ion pair, quantitative ion pair and collision gas energy are shown in Table 3 below.

[0065] Table 3

[0066] Note: * is the quantitative ion Among them, the characteristic ion chromatogram of lincomycin standard is as follows Figure 1 As shown, where: Figure 1 A) Representative characteristic ion mass chromatogram of lincomycin standard. Figure 1 B) Represents the characteristic ion accuracy ratio of lincomycin standard. Figure 1 C) Representative characteristic daughter ion fragmentation pattern of lincomycin standard.

[0067] The characteristic ion chromatogram of Tilmicosin standard is as follows Figure 2 As shown, where: Figure 2 A) Characteristic ion mass chromatogram of tilmicosin standard. Figure 2 B) Represents the characteristic ion accuracy ratio of tilmicosin standard. Figure 2 C) Representative characteristic daughter ion fragmentation pattern of tilmicosin standard.

[0068] The characteristic ion chromatogram of erythromycin standard is as follows Figure 3 As shown, where: Figure 3 A) Representative characteristic ion mass chromatogram of erythromycin standard. Figure 3 B) Represents the characteristic ion accuracy ratio of erythromycin standard. Figure 3 C) Representative characteristic daughter ion fragmentation pattern of erythromycin standard.

[0069] The characteristic ion chromatogram of roxithromycin standard is as follows Figure 4 As shown, where: Figure 4 A) Representative characteristic ion mass chromatogram of roxithromycin standard. Figure 4 B) Represents the characteristic ion accuracy ratio of roxithromycin standard. Figure 4 C) Representative characteristic daughter ion fragmentation pattern of roxithromycin standard.

[0070] Depend on Figure 1-4 It can be seen from the characteristic ion accuracy ratio diagram of the four standards that there is overlap in the chromatograms, indicating that the accuracy of the measured substances is relatively high.

[0071] Effect Example 1 (1) In this example, a matrix-matched standard curve was drawn. The specific method is as follows: accurately measure an appropriate amount of mixed standard working solution and add it to 6 portions of extracted and purified air sample residues. Dry it in a 45°C water bath with nitrogen and add the reconstitution solution. Vortex the residue to dissolve it. Prepare a series of matrix-matched mixed standard solutions with concentrations of 0 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL, and 200 ng / mL. Mix well and filter with a microporous filter membrane. Determine by high performance liquid chromatography-tandem mass spectrometry. Use the measured characteristic ion peak area as the ordinate and the corresponding standard solution concentration as the abscissa to draw a standard curve and calculate the regression equation and correlation coefficient.

[0072] Among them, the standard curve of lincomycin is as follows Figure 5 The standard curve of tilmicosin is shown in Figure 6 The standard curve of erythromycin is shown in Figure 7 shown.

[0073] (2) The method for determining the spike recovery rate in the present invention is as follows: a blank sample is taken for each matrix, and 20 μL, 50 μL or 100 μL of a mixed standard of lincomycin, tilmicosin and erythromycin at a concentration of 1 μg / mL (referred to as the external standard) is added. After sample pretreatment, the concentration of the corresponding standard on the instrument is 20 ng / mL, 50 ng / mL or 100 ng / mL. After detection under the preset test conditions, the spike recovery rate is calculated.

[0074] Effect Example 2 In order to investigate the effect of the amount of added external standard solution on the spike recovery rate, the following experiment was conducted: On the basis of using honeysuckle as the matrix in Example 1, only "adding 100 μL of roxithromycin diluted to 1 μg / mL as the internal standard" was replaced by "adding 100 μL of roxithromycin diluted to 1 μg / mL as the internal standard, and adding 50 μL of a mixed standard of lincomycin, tilmicosin, and erythromycin at a concentration of 1 μg / mL as the external standard". The other parameters were the same as in Example 1. The final test solution prepared was recorded as test solution 1-50; On the basis of using honeysuckle as the matrix in Example 1, only "adding 100 μL of roxithromycin diluted to 1 μg / mL as the internal standard" was replaced by "adding 100 μL of roxithromycin diluted to 1 μg / mL as the internal standard, and adding 100 μL of a mixed standard of lincomycin, tilmicosin, and erythromycin at a concentration of 1 μg / mL as the external standard". The remaining parameters were the same as in Example 1. The final test solution prepared was recorded as test solution 1-100; In Example 2, based on the chrysanthemum matrix, only "adding 100 μL of roxithromycin diluted to 1 μg / mL as the internal standard" was replaced by "adding 100 μL of roxithromycin diluted to 1 μg / mL as the internal standard, and adding 50 μL of a mixed standard of lincomycin, tilmicosin, and erythromycin at a concentration of 1 μg / mL as the external standard". The remaining parameters were the same as in Example 2. The final test solution was recorded as test solution 2-50. In Example 2, based on the chrysanthemum matrix, only "adding 100 μL of roxithromycin diluted to 1 μg / mL as the internal standard" was replaced by "adding 100 μL of roxithromycin diluted to 1 μg / mL as the internal standard, and adding 100 μL of a mixed standard of lincomycin, tilmicosin, and erythromycin at a concentration of 1 μg / mL as the external standard." The remaining parameters were the same as in Example 2. The final test solution was recorded as test solution 2-100. The test solution 1-50, the test solution 1-100, the test solution 2-50 and the test solution 2-100 were respectively injected into the high performance liquid chromatography mass spectrometer and measured under the measurement conditions described in the verification example. The spiked recovery results of lincomycin, tilmicosin and erythromycin in different test solutions are shown in Table 4. In addition, according to the sample processing method for the detection of macrolide antibiotics in plant-derived matrix samples provided in Example 3-4, the test solution prepared after adding the external standard can also achieve the same technical effect as the test solution 1-50 and the test solution 2-50, which will not be repeated here.

[0075] Table 4

[0076] As shown in Table 4, in the 50 μL external standard addition group, the spiked recoveries of lincomycin, tilmicosin and erythromycin in the honeysuckle or chrysanthemum matrix were between 81.52% and 100.22%; in the 100 μL external standard addition group, the spiked recoveries of lincomycin, tilmicosin and erythromycin were between 92.00% and 109.37%. In view of the complex composition and high impurity content of the plant-derived matrix, the current standards and specifications for pesticide residue detection clearly state that a spiked recovery of 70% to 120% is considered to meet the requirements. In the detection method provided by the present invention, the spiked recoveries of lincomycin, tilmicosin and erythromycin are generally within the range of 80% to 110%, indicating that the accuracy of the method meets the detection requirements.

[0077] Effect Example 3 In the process of preparing the test solution in Examples 1-2 and Comparative Examples 1-8, after adding the internal standard, 50 μL of a mixed external standard of lincomycin, tilmicosin and erythromycin at a concentration of 1 μg / mL was added, and the remaining steps were unchanged. The corresponding test solutions 1-50, test solutions 2-50, test solution pair 1'-test solution pair 8' were prepared. Each group of test solutions was measured twice, and the spiked recovery rates of lincomycin, tilmicosin and erythromycin were determined according to the method provided in the verification example, and analyzed. Among them, the determination results of the spiked recovery rates of the corresponding antibiotics in different test solutions are shown in Tables 5-6. Among them, Table 5 is the determination results of the spiked recovery rates in the corresponding test solutions prepared with honeysuckle as the matrix; Table 6 is the determination results of the spiked recovery rates in the corresponding test solutions prepared with chrysanthemum as the matrix.

[0078] Table 5

[0079] Table 6

[0080] As can be seen from Tables 5-6, in the experiment of determining the spiked recovery of lincomycin, tilmicosin and erythromycin by adding 50 μL of mixed external standard to the honeysuckle or chrysanthemum matrix, when other parameters were the same, compared with other groups, after treatment with the adsorbent comprising O-terminated octadecyl bonded silica gel provided by Examples 1-2 of the present invention, the average spiked recovery of the three antibiotics lincomycin, tilmicosin and erythromycin was closer to 100%, and the test solution obtained in this way had better detection accuracy.

[0081] Among them, after replacing the "O-terminated octadecyl bonded silica gel" in the adsorbent described in Example 1-2 with conventional unterminated octadecyl bonded silica gel (ODS C18), the response ratio of the external standard to the internal standard became higher, which made the spiked recovery rate value higher. After treatment with the replaced adsorbent, the average spiked recovery rate of the test solution finally obtained deviated from 100% to a greater extent, and the accuracy was relatively reduced.

[0082] When the "O-capped octadecyl bonded silica gel" in the adsorbent described in Examples 1-2 was replaced with an equal amount of "graphite carbon," "neutral alumina," or "acidic alumina," the internal standard response was too low during detection. In particular, the response to tilmicosin and erythromycin approached zero, resulting in an external standard response ratio of essentially zero. The final concentration value could not be accurately displayed.

[0083] During the experiment, the adsorbent is a key factor affecting sample processing, so the choice of adsorbent type and combination is crucial. Replacing the O-terminated octadecyl bonded silica gel in the adsorbent protected by the present invention with graphite carbon resulted in a higher removal rate of pigments from the sample. However, the graphite carbon also adsorbed cyclic planar structures in the sample, resulting in a low spike recovery rate.

[0084] When the O-terminated octadecyl bonded silica gel is replaced with alumina, the released aluminum ions can bind to the groups in the analyte, potentially generating or precipitating metal complex groups or chelating with mineral ions to form insoluble salts. The addition of alumina can result in low spike recovery rates or even failure to detect the analyte.

[0085] Effect Example 4 This example investigates the effect of the type of anhydrous sulfate in the mixed inorganic salt and adsorbent on the detection accuracy of the test solution obtained after sample treatment.

[0086] On the basis of the sample processing method for detecting macrolide antibiotics in plant-derived matrix samples provided in Example 1, after the sample processing step (3) of "adding 100 μL of roxithromycin diluted to 1 μg / mL as an internal standard", 20, 50 and 100 μL of a mixed standard of lincomycin, tilmicosin and erythromycin with a concentration of 1 μg / mL were added respectively; the remaining steps and parameters were the same as in Example 1, and finally the test solutions were prepared, and the obtained test solutions 1 were respectively recorded as test solution 1-20, test solution 1-50 and test solution 1-100.

[0087] On the basis of the sample processing method for detecting macrolide antibiotics in plant-derived matrix samples provided in Example 2, after the step (3) of sample processing "adding 100 μL of roxithromycin diluted to 1 μg / mL as an internal standard", 20, 50, and 100 μL of a mixed standard of lincomycin, tilmicosin, and erythromycin at a concentration of 1 μg / mL were added, respectively; the remaining steps and parameters were the same as in Example 2, and finally the test solutions were prepared, which were respectively recorded as test solution 2-20, test solution 2-50, and test solution 2-100.

[0088] According to the method provided in the verification example, the spiked recoveries of lincomycin, tilmicosin and erythromycin in the above-mentioned test solutions 1-20 to test solutions 2-100, 9 series of test solutions and 10 series of test solutions were respectively measured and analyzed. Among them, the determination results of the spiked recoveries of the corresponding antibiotics in different test solutions are shown in Tables 7-8. Among them, Table 7 is the determination results of the spiked recoveries of test solutions 1-20 to test solutions 2-100; Table 8 is the determination results of the spiked recoveries of 9 series of test solutions and 10 series of test solutions. Among them, the liquid chromatogram of test solution 1-50 is shown in Figure 8 The liquid chromatogram of the test solution 2-50 is shown as Figure 9 shown.

[0089] Table 7

[0090] Table 8

[0091] As can be seen from Tables 7-8, in the experiment of determining the spiked recovery of lincomycin, tilmicosin and erythromycin by adding 100 μL of internal standard and 20 μL-100 μL of mixed external standard to the honeysuckle or chrysanthemum matrix, when other parameters are the same, compared with the mixed inorganic salt or adsorbent containing anhydrous sodium sulfate in the comparative example 9-10 group, after treatment with the mixed inorganic salt or adsorbent containing anhydrous magnesium sulfate provided by Examples 1-2 of the present invention, the average spiked recovery of the three antibiotics lincomycin, tilmicosin and erythromycin is closer to 100%, and the test solution obtained in this way has better detection accuracy. The reason may be that the original anhydrous magnesium sulfate in the mixed inorganic salt initially removes excess water and macromolecular substances in the original sample and does not react with the test substance, nor does it affect the properties of the extract. After being replaced by anhydrous sodium sulfate, sodium ions will be more active under acidic conditions and may react with ions under acidic conditions. Due to the complexity of the matrix, the detection of lincomycin, tilmicosin and erythromycin in the final test solution is affected.

[0092] The above-described embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A sample processing method for detecting macrolide antibiotics in plant-derived matrix samples, characterized in that: The sample processing method comprises the following steps: The plant-derived matrix sample is mixed with a buffer solution, acidified acetonitrile is added for extraction, and then mixed inorganic salts are added to the extract, solid-liquid separation is performed, and the resulting liquid phase is added to an adsorbent for adsorption, solid-liquid separation is performed again, and the liquid phase is obtained to obtain the treated plant-derived matrix sample; The adsorbent includes anhydrous magnesium sulfate, aliphatic diamine derivatives and O-terminated octadecyl bonded silica gel.

2. The sample processing method for detecting macrolide antibiotics in a plant-derived matrix sample according to claim 1, characterized in that: The mass ratio of the anhydrous magnesium sulfate, the aliphatic diamine derivative and the O-terminated octadecyl bonded silica gel is 16-20:0.8-1.2:0.8-1.

2.

3. The sample processing method for detecting macrolide antibiotics in a plant-derived matrix sample according to claim 2, characterized in that: The mass ratio of the plant-derived matrix sample to the adsorbent is 0.4-0.6:1.8-2.

2.

4. The sample processing method for detecting macrolide antibiotics in a plant-derived matrix sample according to claim 1, wherein The macrolide antibiotics include at least one of lincomycin, tilmicosin, roxithromycin or erythromycin; and / or The mixed inorganic salt includes strong acid salt and anhydrous sulfate.

5. The sample processing method for detecting macrolide antibiotics in a plant-derived matrix sample according to claim 4, characterized in that: The mass ratio of the strong acid salt to the anhydrous sulfate is 1.5-2.5:3.5-4.5; and / or The strong acid salt comprises sodium chloride; and / or The anhydrous sulfate includes anhydrous magnesium sulfate.

6. The sample processing method for detecting macrolide antibiotics in a plant-derived matrix sample according to claim 1, characterized in that: The aliphatic diamine derivatives include N-propylethylenediamine.

7. The sample processing method for detecting macrolide antibiotics in a plant-derived matrix sample according to claim 1, characterized in that: The volume ratio of the organic weak acid to acetonitrile in the acidified acetonitrile is 0.08-0.12:99.88-99.

92.

8. The sample processing method for detecting macrolide antibiotics in a plant-derived matrix sample according to claim 1, wherein: After the adsorbent is added for adsorption, the solid and liquid are separated, the obtained liquid phase is dried, and then a formic acid aqueous solution-methanol mixture is added for redissolution, and the sample is filtered to obtain a treated plant-derived matrix sample.

9. The sample processing method for detecting macrolide antibiotics in a plant-derived matrix sample according to claim 8, characterized in that: The volume proportion of formic acid in the formic acid aqueous solution is 0.18%-2.4%; and / or The volume ratio of the formic acid aqueous solution to methanol is (7.8-8.2): (1.8-2.2).

10. The sample processing method for detecting macrolide antibiotics in a plant-derived matrix sample according to claim 1, characterized in that: The plant-derived matrix sample includes at least one of honeysuckle or chrysanthemum.

Images